Toll-Like Receptors in the Pathogenesis of Autoimmune Diseases

2015 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2015, 5(Suppl 1), 605-614 doi: 10.15171/apb.2015.082 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Overview on TLRs
The innate immune system, an organism's first line of defense against invading pathogens, consists of different molecules and cells that work in conjunction with the adaptive immune system to maintain physiological homeostasis of the host and to protect it against potentially pathogenic organisms. 1Recognition of a wide range of molecular structures that presents in different microorganisms is dependent on a diverse set of germ line encoded receptors, termed pattern recognition receptors (PRRs). 2 These receptors are expressed by a variety type of cellsespecially the innate immune system cells, such as dendritic cells (DCs) and macrophagesand they act to sense danger or damage signals.The PRR ligands comprise conserved microbial structures, named pathogen-associated molecular patterns (PAMPs), such as viral and bacterial nucleic acids, lipopolysaccharide (LPS) and flagellin.In addition, PRRs can sense damageassociated molecular patterns (DAMPs, endogenous danger signals from dead and dying cells), such as saturated fatty acids and amyloid β. 3 Toll-like receptors (TLRs), the best characterized PRRs, were first reported in humans in 1998. 3,46][7] Each TLR is able to recognize a particular molecular pattern.For example, TLR2, 4, 5, 6 and 11 bind to bacterial membrane-associated molecules such as LPS, lipoprotein and peptidoglycan whereas TLR3, 7, 8 and 9 sense viral and bacterial or endogenous nucleic acids, including ssRNA, dsRNA, and unmethylated cytosine phosphate guanine (CpG) -containing DNA (Table 1).Also, TLRs can be classified based on their localization in the cell so that TLR1, 2, 4, 5 and 6 are expressed on the cell membrane, whereas TLR3, 7, 8 and 9 are localized mainly in the endosomal compartment. 8Triggering of TLRs upon ligand binding results in signaling events that lead to the expression of some immune response genes, including inflammatory cytokines, stimulatory immune cytokines, chemokines, and costimulatory molecules (Figure 1), which augment the killing of pathogens and initiates the process of developing acquired immunity. 4hen TLRs are stimulated by their ligands, they recruit downstream adaptor molecules, such as myeloid Toll/interleukin (IL) -1 receptor (TIR) -domain-containing adaptor-inducing interferon-β (TRIF), TRIF-related adaptor molecule (TRAM) and differentiation primaryresponse protein 88 (MyD88) which trigger other downstream molecules leading to the activation of signaling cascades that converge at the nuclear factor-kB (NF-kB), interferon (IFN) response factors (IRFs) and mitogen-activated protein (MAP) kinases.These transcription factors induce the transcription of some proinflammatory agents, such as interleukin (IL) -6, IL-12, IL-23, and tumor necrosis factor α (TNF-α).The production and secretion of these molecules counters the threat posed by microbes and helps activate other immunological components.AP1, activator protein 1; ATF, activating transcription factor; ERK, extracellular signal-regulated kinase; IKK, inhibitor of kappa light polypeptide gene enhancer in B-cell kinase; IRAK, IL-1 receptor-associated kinase; JNK, c-Jun N-terminal kinase; MKK, MAPK kinase; RIP1, receptor interacting protein 1; TAB, transforming growth factor-b-activated kinase 1/MAP3K7-binding protein; TAK, transforming growth factor-activated kinase; TRAF, tumor necrosis factor receptor-associated factor.

TLRs and autoimmunity
Autoimmunity is emerged by several coincident mechanisms that relate to the presence of auto-reactive immune cell subsets and loss of immunological tolerance. 10Loss of tolerance during central and peripheral differentiation of the adaptive immune response may lead to uncontrolled activation of self-reactive B and T cells which induce autoimmunity assisted by the cells of the innate immunity.TLR expression has been found in various types of immune cells, including T cells, B cells, different subsets of dendritic cells, monocytes, macrophages, neutrophils, eosinophils, mast cells, and epithelial cells (Table 2). 8,11,19,24TLR signaling plays an essential role in the Toll-like receptors in autoimmunity Advanced Pharmaceutical Bulletin, 2015, 5(Suppl 1), 605-614 activation of the adaptive immune system by inducing the production of pro-inflammatory cytokines and upregulating costimulatory molecules of antigen presenting cells (APCs). 7Considering the role of TLRs as a critical link between the innate and the adaptive immune responses, the idea has created that continuous activation or dysregulation of TLR signaling might contribute to the pathogenesis of autoimmunity. 25,26

Role of TLRs on B cells function in autoimmunity
Systemic autoimmune diseases are frequently associated with the production of autoantibodies that react with nuclear or cytosolic cellular components.These autoantibodies have an important role in the immunopathogenesis of various autoimmune diseases. 31 key finding which brought the logical link between the TLRs that recognize nucleic acids and the production of antibodies (Abs) that recognizes nucleic acids involved studies on AM14 transgenic mice.The B cells of these mice express specific Abs for self-IgG, also known as rheumatoid factor.32 In this study, anti-nucleosomal IgG was mixed well with cell lysates to induce B cell proliferation in transgenic B cells.33 Furthermore, this stimulation was DNase sensitive and depends on MyD88, and TLR9 inhibitory oligonucleotides could abrogate the response of the auto-reactive B cells.These observations provided the first report of a synergistic effect between a DNA-binding TLR and a BCR that binds and internalizes DNA-containing complexes.More support of the role of DNA as a stimulus of auto-reactive B cells was shown by using transgenic 3H9 B cells that have a specific BCR for DNA.34 With this system, CpGassociated proliferation was also sensitive to TLRinhibiting oligonucleotides, confirming the idea that combined signals via TLRs and the BCR provide a mechanism to activate auto-reactive B cells. 31 Using an animal model of autoimmunity making by an anti-DNA BCR transgene and homozygous deficiency of the FcγR IIB, an inhibitory receptor, it was shown that isotype switching of auto-reactive B cells to the IgG2a and 2b subclasses requires TLR9 and MyD88 signaling; accordingly, deficiency in TLR9 or MyD88 resulted in reduced pathology and mortality in this model.35 As well, B cells containing the Y-linked autoimmune accelerator (Yaa), a genetic modifier, have an intrinsic tendency toward the production of more pathogenic anti-nucleolar autoantibodies. Anlysis of the Yaa locus showed a duplication of the TLR7 gene. 36Also, the importance of TLR7 role as a coreceptor to activate auto-reactive B cells are more supported by a study using a BCR knockin mouse with specificity for a number of nuclear antigens.Breakage of tolerance and autoantibody production in this mouse was largely dependent on TLR7.37 Finally, by analyzing chimeric mice, where deficiency in TLR9 is restricted to the B cell lineage, suggests that TLR9 promotes the activation and differentiation of anti-DNA plasmablasts in murine lupus via a B cell-intrinsic mechanism.38,39 Also, TLR4 signaling directs peripheral B cells to rapidly locate within the lymph nodes (LNs) and the splenic white pulp.So, it probably does by enhancing chemokine receptor expression, increasing the expression of L-selectin molecules, and biasing the signaling pathway toward heightened responsiveness.The TLR4-activated cells predominantly reside in the center of the LN follicle where they can directly interact with each other.If an LN follicle contains a germinal center, exposure to a TLR4 ligand will promote the entrance of stimulated B cells into the dark zone.Signals from TLR response provide a mechanism that augments the polyclonal expansion of the B cells which happens after exposure to infectious agents and during the course of autoimmune disorders.40 In humans, the peripheral dendritic cells (pDCs) expressing TLR-9 recognize bacterial and viral nucleic acids.So, the activation of pDCs through TLR-9 leads to production and release of type I IFN (IFN-α, IFN-β) which enhance humoral immunity.In fact, IFN-α enables B cells to undergo isotype-switching and mature into antibody-secreting cells.41 Also, TLR-9 stimulation with CpG_oligodeoxynucleotide (ODN) induces naive B cell maturation into memory cells, as demonstrated by the AID mRNA expression, induction of CD27 and IgG production.On the other hand, IFN-α produced by pDCs amplifies the inductive effect of CpG-ODN on naive B cells activation and on Ig production through a mechanism involving TLR-9/MyD88-dependent signaling (by an up-regulation of MyD88 expression).Thus, TLR-9 signaling plays a direct role on naive B cells, comprising Ig production with a possible risk for autoimmunity, and an indirect role on pDCs, comprising IFN-α production, which boost IgM production, further amplifies the susceptibility to autoimmune disorders.42

Role of TLRs on T cells function in autoimmunity
Researches showed that T helper 17 (TH17) and TH1 cells or IL 17+ IFNγ+ CD4+ T cells may have pathogenic roles in chronic inflammatory and autoimmune diseases.Before the discovery of TH17 cells, it was believed that TH1 cells were the primary pathogenic cell population in T cell-mediated autoimmune disorders.4][45] It was subsequently shown that IL 17-/-mice have a reduced susceptibility to EAE, 46 and that EAE can be induced in naive mice following the transfer of myelin-specific TH17 cells that are isolated from mice with EAE and expanded in vitro using IL 23. 47 It has also been reported that the transfer of auto-reactive TH1 cells into naive recipients induced experimental autoimmune uveitis (EAU) in this group, and this was not inhibited by treatment with specific anti-IL-17 antibodies. 48Finally, CD4+ T cells that express both IL 17 and IFNγ have been found in the gut of patients with Crohn's disease. 49t is well established that TLR signaling in innate immune cells can indirectly promote T cell differentiation and proliferation through DC maturation and regulatory cytokine production.TLR3, TLR4 or TLR9 ligands induce MyD88 dependent production of cytokines from DCs that promotes the differentiation of TH17 cells. 50Moreover, the TLR4 ligand LPS promotes IL 17 production by antigen-specific memory T cells in mice through the induction of IL 1 and IL 23 production by DCs. 51T cells also express TLRs, and evidence is showing that TLR signaling in T cells can promote the cytokine production or regulate their function.

Role of TLRs in regulatory T cells function in autoimmunity
Regulatory CD4+ CD25+ T (Treg) cells with the ability to suppress host immune responses against self-or nonself antigens have significant roles in the processes of autoimmunity.Interestingly, new evidence proposes that TLR signaling may directly or indirectly regulate the immunosuppressive function of CD4+ CD25+ Treg cells in immune responses.TLR signaling may shift the balance between CD4+ T-helper cells and Treg cells, and finally influence the outcome of the immune response. 11he effect of TLR signaling on Treg cells suppression experiments indicated that TLR ligands such as LPS and unmethylated CpG DNA motifs (which bind TLR4 and TLR9, respectively) allowed T cells to proliferate even in the presence of Tregs.It is now supposed that CpG-DNA can directly costimulate T-cell proliferation and also can make T cells resistant to Treg suppression in a MyD88dependent manner. 52,53Ligation of TLR2 has been reported to confer resistance to suppression of Treg, in part by increasing IL-2 production. 54In the other hand, following the extension of TLR2-driven proliferation, Treg suppression appeared to be enhanced. 27Also ligation of flagellin to TLR5 has been shown to increase Treg suppressive capacity. 19In contrast, triggering of TLR8 with nucleic acid ligands has been shown to directly impair Treg function. 21Thus, Treg suppression can be either boosted or attenuated by distinct TLR ligands. 55

Role of TLRs in the pathogenesis of autoimmune diseases Systemic lupus erythematosus (SLE)
Increasingly, data from both murine and human studies have implicated TLR activation in the pathogenesis of SLE.SLE is a systemic autoimmune disease characterized by the production of autoantibodies. 56The patient sera contain endogenous ligands for TLRs, particularly the nucleic acid binding TLRs including TLR7, TLR8 and TLR9. 57,58In patients with SLE, autoreactive cells are producing large quantities of autoantibodies against self-nuclear antigens which make immune complexes with self-nucleic acids and present in SLE serum.Since self-RNA and self-DNA in the form of protein complexes can act as TLR7 and TLR9 ligands, stimulation of TLRs is suggested as an additional signal contributing to activate and/or modulation of the aberrant adaptive immune response.Data from researches on mouse models suggest a pathogenic role for TLR7 signaling and a protective role for TLR9 signaling in the pathogenesis of SLE. 59eumatoid arthritis (RA) RA is a chronic inflammatory autoimmune disease characterized by the progressive and irreversible destruction of joints. 60Some of the endogenous TLR ligands can be found in arthritic joints.Recently it was shown that RNA released from necrotic synovial fluid cells of RA patients can activate TLR3 on RA synovial fibroblasts. 61The presence of endogenous TLR4 ligands such as fibronectin fragments and heat-shock proteins (HSPs) has also been demonstrated in rheumatoid synovium, [62][63][64] and it has been reported that rheumatoid synovial fibroblast-like cells synthesize extra domain A (ED-A) -containing fibronectin. 65In addition, serum and synovial fluid of RA patients can activate a TLR4expressing ovary cell line of Chinese hamster, suggesting the presence of TLR4-activating substances in RA serum and joints. 25,66ltiple Sclerosis (MS) MS is an autoimmune disease in which central nervous system (CNS) lesions result from the perivascular immune cell infiltration associated with damage to myelin, oligodendrocytes and neurons.8][69] Ligation of TLR2 and TLR4 of DCs with endogenous ligands such as high mobility group box 1 (HMGB1) induces the production of IL1, IL6 and IL12 that stimulate the differentiation of naive T cells into Th1 and Th17 cells, which secrete INFγ and IL17 respectively.INFγ/IL17-producing T cells facilitate leukocyte migration through the blood-brain barrier and contribute to CNS damage. 70

Experimental autoimmune encephalomyelitis (EAE)
EAE is a family of models of autoimmune CNS damage induced by the immunization of experimental animals with CNS components (either an extract, purified protein or a peptide), usually in the presence of an adjuvant, 71 which in turn promote pathogenic auto-reactive T cell responses.Studies showed that mice deficient in the adaptor protein MyD88 is resistant to EAE, which associated with decreased IL 6 and IL 23 production by DCs and decreased IFNγ and IL 17 production by T cells. 72,73This suggests that innate immune responses that are initiated through TLR or IL 1R signaling are required for the induction of experimental autoimmunity.Mice with a lymphoid cell-specific TLR2 deficiency is less susceptible to EAE and have reduced Th17 responses compared with wild-type controls. 74Also, it has been reported that parenteral injection of polyinosinic-polycytidylic acid (poly I: C), a TLR3 ligand, suppressed relapsing-remitting EAE. 75ollectively, these findings indicate that TLR and IL 1R signaling is crucial for the induction of EAE.Moreover, depending on the stage of the disease, some TLRs (including TLR2 and TLR9) may have pro-inflammatory functions, whereas others (such as TLR3 and TLR9) may also have a regulatory role in the EAE process. 3

TLRs as drug targets for autoimmunity
Since the bulk of the data suggests that the TLR pathway plays a key role in autoimmune disease pathogenesis, targeting TLRs and their signaling pathways may provide an effective therapeutic approach for certain autoimmune diseases.Since there are several proteins involved in TLR signaling, there are various targets that may be utilized for potential drugs (Table 3).

Conclusion
TLR molecules and their downstream signaling pathways play a critical role in activating innate and adaptive immune cells.Their specific role in modulating immunity also interferes with the mechanisms that maintain tolerance in the host.Thus, aberrantly expressed or activated of TLRs can contribute to the loss of tolerance by multiple mechanisms.Given that this pathway is important in several autoimmune diseases, this axis constitutes an attractive target for therapeutic intervention.

Figure 1 . 2 .
Figure 1.Overview of the Toll-like receptor signaling pathway

Table 3 .
Toll-like receptor targeting drugs Drug Mechanism of function References IRS 661 Block signaling via TLR-7 and inhibit the production of IFN-α by human plasmacytoid dendritic cells (pDC) 2,76-81 Nucleic acid-binding polymers Recognize ssRNA, dsRNA or hypomethylated DNA, inhibit nucleic acid-mediated activation specifically designed to inhibit MyD88 dimerization, interfere with the recruitment of IRAK1 and IRAK4 by MyD88, resulting in the inhibition of IL-1βmediated activation of NF-κB and IL-6.Suppressed B cell proliferation and differentiation into plasma 88 Compound 4a Interfere with the interaction between MyD88 and IL-1R at the TIR domains 89 RO0884 Dual inhibitor of IRAK1 and IRAK4, block pro-inflammatory cytokine production 90 Synthetic oligo deoxynucleotides with immunoregulatory sequences (IRS)

Table 1 .
Toll-like receptors and their ligands, adaptor usage,